Noi/NOTES/2014-2-13

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scRRBS trial experiment #1[edit]

  • Link to calendar: [[1]]

2014-02-12[edit]

  • Have nuclei from Script
  • Rui helped to spin down the cell, resuspend with buffer (0.6%BSA/PBS), and count cell concentration.
  • There are two tubes with total volume ~15ul left for me.
    • ~700 cells/ul
    • ~1000 cells/ul
  • Dilute nuclei from 700 nuclei/mL tube to 100 nuclei/ul and 10 nuclei/ul and put 1ul to each well with 4ul lysis buffer (8 strips).
  • I used sorted buffer as NTC
Layout[edit]
Well #1 Well #2 Well #3 Well #4 Well #5 Well #6 Well #7 Well #8
NTC 10 nuclei 100 nuclei 1000 nuclei NTC 10 nuclei 100 nuclei 1000 nuclei

2014-02-13[edit]

  • I plan to do experiment on 1 strip tube

Things to prepare[edit]

- Dilute protease to 10mg/mL (follow reagent prep [[2]])
- Dilute unmethylated lambda DNA to 120fg/ul (follow reagent prep)
- Mix protease and unmethylated lambda DNA 1:1 ratio (5ul + 5ul). This will be used 1ul for cell lysis.

Cell lysis[edit]

- Thaw nuclei from -20C & spin down
- Add 1ul of protease/unmethylated lambda DNA mix
- Incubate at 50C for 3hr
- Heat inactivate at 75C for 30min


Msp I digestion[edit]

- Aliquot 10X Tango buffer 60ul/tube (3strips)
Components Volume (ul) 9x rxn mix
Lysed nuclei 5.00 0.00
10X Tango buffer 2.00 18.00
MspI 0.90 8.10
H2O 10.10 90.90
Total 18.00 117.00
- Aliquot 13ul to each tube
- Incubate at 37C for 3hr


End-repair/dA-tailing[edit]

- Prepare dA/dC/dG (20mM:2mM:2mM) mix (follow reagent prep)
- The new set of dNTP solution has not come in yet, so I used the old set in common reagent box.
Components Volume (ul)
MspI digested reaction 18.00
dA:dC:dG mix (20mM, 2mM,2mM) 1.00
Klenow fragment exo- 1.00
Total 20.00
- Add 1ul of dA/dC/dG mix
- Add 1ul of Klenow fragment exo-
- Incubate at 30C for 15min
- Heat inactivate at 75C for 10 min**. (This step was not mentioned in paper. I may skip it next time)


Methylated adapter ligation[edit]

- Prepare methylated adapter: 5000 ng requires 10ul adapter, so 1ng requires -->(1ng * 10ul)/5000ng = 0.002ul
' 1x rxn (ul) 1000x rxn (ul)
Methylated adapter 0.002 2.00
H2O 0.998 998.00
Total 1.000 1000.00


Components Volume (ul) 9x rxn mix
dA-tailed reaction 20.00 0.00
10X Tango buffer 0.50 4.50
HC T4 DNA ligase (30units/ul) 1.00 9.00
10mM ATP 1.25 11.25
50% PEG 4000 1.25 11.25
Total 24.00 36.00
- Add 1ul of diluted methylated adapter
- Add 4ul of ligation reaction mix
- Mix by pipetting and spin down
- Incubate at 22C for 1hr
  • After ligation, I want to test if ligation work. One set of adapter ligated DNA was column purified with minElute QIAGEN column (followed standard protocol), and PCR was performed without bisulfite conversion. Another set was bisulfite treated.


Bisulfite conversion[edit]

Yeast tRNA preparation[edit]

Components Initial conc. Unit Diluted conc. Unit Volume for 100ul Unit H2O (ul) Total volume (ul)
Yeast tRNA 10.00 mg/mL or ug/ul or X10^3ng/uL 1.00 ug/ul 10.00 ul 90.00 100.00
Yeast tRNA 1.00 ug/ul or X10^3ng/ul 2.00 ng/ul 2.00 ul 998.00 1000.00
- Follow the protocol provided by manufacturer: File:Methylcode bisulfite man.pdf
- Mix 25ul of DNA sample with 162.5ul of CT Conversion Reagent
- 98°C for 10 minutes (DNA denaturation)
- 64°C for 2.5 hours (Bisulfite conversion)
- 4°C storage for up to 20 hours or continue to desulfonation
- Bind DNA to column by mixing with 750ul Binding Buffer and 5ul of 2ng/ul tRNA as carrier. Spin down 14,000rpm for 30sec
- Wash with 100ul Wash buffer. Spin down 14,000rpm for 30sec
- Incubate with 200ul of Desulphonation Buffer for 15min. Spin down 14,000rpm for 30sec
- Wash column with 200ul Wash Buffer. Spin down 14,000rpm for 30sec. Discard spnt.
- Wash the column with 200ul Wash Buffer. Spin down 14,000rpm for minsec
- Elute converted DNA with 13ul Elution Buffer. This should have ~10ul DNA left for PCR


PCR amplification[edit]

  • I HAVE CHANGED ANNEALING TEMPERATURE FROM 60C to 65C IN BOTH 1ST and 2ND ROUND OF AMPLIFICATION

1st round amplification[edit]

Components Conc unit Final conc. unit Volume (ul) 10 rxn mix
Bis-cvt DNA 10.00 0.00
10X Reaction buffer 10 X 1 X 5.00 50.00
dNTP mix 10 mM 0.2 mM 1.00 10.00
PCR_F 10 uM 0.1 uM 0.50 5.00
PCR_N2.Ind2 10 uM 0.1 uM 0.50 5.00
50X SYBG 50 X 1 X 1.00 10.00
PfuTurbo Cx 2.5 Unit/ul 1 unit 0.40 4.00
H2O 31.60 316.00
- Aliquot 40ul, add 20ul of DNA template
- 95C for 2min --> [95C for 20sec -> 65C for 30sec -> 72C for 1min] X 25 cycles --> 72C for 2min
- Perform two rounds AMPure bead purification

AMPure bead purification[edit]

- Add 50ul AMPure bead. Mix by pipetting 10x
- Sit for 8min
- Transfer to sit on magnet for 3min
- Wash twice with 150ul freshly prepared 80% EtOH
- Dry the bead for 3-5min
- Resuspend with 40 H2O
- Add 40ul AMPure bead. Mix by pipetting 10x (Note that, AMPure beads in the first round were saved and combined with the second round beads). In the future, I may only add binding buffer, 20%PEG/NaCl, in the second round of purification.
- Sit for 8min
- Transfer to sit on magnet for 3min
- Wash twice with 150ul freshly prepared 80% EtOH
- Dry the bead for 3-5min
- Resuspend with 25ul H2O

2nd round amplification[edit]

Components Conc unit Final conc. unit Volume (ul) 10 rxn mix
Purified 1st round DNA 25.00 0.00
5X Phusion HF buffer 5 X 1 X 10.00 100.00
dNTP mix 10 mM 0.2 mM 1.00 10.00
PCR_F 10 uM 0.1 uM 0.50 5.00
PCR_N2.Ind2 10 uM 0.1 uM 0.50 5.00
50X SYBR 50 X 0.8 X 0.80 8.00
Phusion HF 2 unit/ul 0.50 5.00
H2O 11.70 117.00
- Aliquot 25ul, add 25ul of bead purified 1st round PCR
- 98C for 2min --> [98C for 10sec -> 60C for 30sec -> 72C for 1min] X 22 cycles --> 72C for 2min
- I monitored qPCR and stopped reaction at 22cycles

PAGE analysis[edit]

- Loaded 3ul of PCR product in 6% TBE gel
File:ZhangLab 2 2014-02-14 17hr 42min scRRBS 2ndround-3ulPCR.jpg


DISCUSSION WILL BE ADDED

  • Continued on: [[3]]